Methyl α-D-mannopyranoside

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay (reference 7.6.1-1).

The test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells (refrence 7.6.1-2).

The test item was determined to be non-mutagenic in a HPRT assay using V79 cells of Chinese hamster (reference 7.6.1-3).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-08 to 2021-06-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line (from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line.
- Normal cell cycle time (negative control): see below

For cell lines:
- Absence of Mycoplasma contamination: Before freezing each batch is screened for mycoplasm contamination.
- Methods for maintenance in cell culture: Thawed stock cultures are propagated at 37 °C in 175 cm2 plastic flasks. About 5 x 105 cells per flask are seeded in 30 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium is supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells are sub-cultured twice a week. All incubations are done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
- Cell cycle length, doubling time or proliferation index: doubling time of V79 cells in stock cultures: approximately 13 hours, determined on May 06, 2011
- Modal number of chromosomes: 22 +/- 2
- Periodically checked for karyotype stability: yes, before freezing

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Please refer to “Methods for maintenance in cell culture” above.

Cytokinesis block (if used):

Cytochalasin B (1.5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9, prepared in study laboratory
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration of S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

With regard to the molecular weight of the test item, 1950 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 12.7 to 1950 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, this preliminary test was designated Experiment I. The experimental part without S9 mix was repeated in Experiment II since the positive control was invalid (too strong cytotoxicity). The experimental part with S9 mix was repeated in Experiment II as confirmatory experiment since a statistically significant increase and dose-dependency were observed in Experiment I. 1950 µg/mL were chosen as top treatment concentration for Experiment II for both experimental parts (4 hours with and without S9 mix). The continuous treatment without S9 mix was performed in Experiment III with the same top dose (1950 µg/mL).

Vehicle / solvent:

- Solvent used: deionised water

- Justification for choice of solvent: The test item was sufficiently soluble in the solvent.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium did not exceed 10 %.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5.0 – 6.0 x 10^5 cells in 25 cm2 plastic flasks
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4 h; Experiment 2: 24 h
- Harvest time after the end of treatment (sampling/recovery times): Experiment 1: 20 h; Experiment 2: none

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis block: Cytochalasin B (1.5 μg/mL), Experiment 1: 20 h; Experiment 2: 24 h
- Methods of slide preparation and staining technique used including the stain used: Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37 °C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) were added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip.
- Number of cells spread and analysed per concentration: duplicate cultures; 500 cells per culture were scored on coded slides for the determination of the CBPI. At least 1000 binucleate cells were scored per culture for micronuclei on coded slides.
- Criteria for scoring micronucleated cells: The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): not applicable


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Rationale for test conditions:

The highest treatment concentration in this study, 1950 µg/mL was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition.
− The results are outside the range of the laboratory historical solvent control data (95 % confidence interval)

Statistics:

Statistical significance will be confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis will be conducted for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.
A linear regression will be performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant influence on pH was observed.
- Data on osmolality: No relevant influence on osmolarity was observed.
- Precipitation and time of the determination: No precipitation of the test item in the culture medium was observed at the end of treatment.
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.

STUDY RESULTS
Either Griseofulvin (7.0 µg/mL), MMC (0.2 µg/mL) or CPA (3.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

- Statistical analysis:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance was considered together.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

- Genotoxicity results
In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed by using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed.

HISTORICAL CONTROL DATA
The historical control data were generated in accordance with the OECD Guideline 487. For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data). For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min max range of the positive controls was applied for the evaluation of acceptability

Summary of results

Exp.	interval	concentration	Proliferation index CBPI	Cytostatsis in %*	Micronucleated cells in %**	95 % ctrl limit
Exposure period 4 hrs without S9 mix
II	24 h	Solvent control ^1	1.93		0.8	0-2.45
		Positive control ^2	1.43	53.7	13.20^s
		637	1.92	0.9	0.65
		1114	1.92	1.2	0.65
		1950	1.90	3.2	0.85
Trend test: p-value 0.740
Exposure period 24 hrs without S9 mix
III	24 h	Solvent control ^1	2.21		1.10	0-1.7
		Positive control ^3	2.30	n.c.	10.90^s
		637	2.07	11.8	0.6
		1114	2.11	8.1	0.6
		1950	2.07	11.2	0.8
Trend test: p-value 0.740
Exposure period 4 hrs with S9 mix
I	24 h	Solvent control ^1#	1.96		0.65	0.01-1.99
		Positive control^4	1.54	43.1	6.20^s
		637^#	1.92	3.8	0.7
		1114^#	1.99	n.c.	0.83
		1950^#	1.96	n.c.	1.15^s
Trend test: p-value 0.040^T
II	24 h	Solvent control ^1	1.87		1.25	0.01-1.99
		Positive control^4	1.34	60.5	22.20
		637	1.87	0.1	0.8
		1114	1.87	n.c.	0.75
		1950	1.85	1.6	0.9
Trend test: p-value 0.418

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

T Trend analysis via linear regression is significant (p ˂ 0.05)

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 Deion. water 10.0 % (v/v)

2 MMC 0.2 µg/mL 3

 Griseofulvin 7.0 µg/mL

4 CPA 3.0 µg/m

 

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.

Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments. Experiment I and II were conducted with and without metabolic activation (S9-mix) with exposure periods of 4 and 24 hours. Experiment III was conducted with out metabolic activation with an exposure period of 24 hours. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1950 µg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-11 to 2021-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster cell line V79 (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time (negative control): doubling time 12 - 16 h

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasm contamination.
- Number of passages: not specified
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3Χ10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly.
- Cell cycle length, doubling time or proliferation index : doubling time 12 - 16 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes. Each master cell stock is checked for karyotype stability.
- Periodically ‘cleansed’ of spontaneous mutants: yes. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air. For details on medium see above.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9, prepared in study laboratory
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration of S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

60.9; 121.9; 243.8; 487.5; 975.0; 1950.0 μg/mL

In the pre-experiment to determine the toxicity of the test item the highest concentration was equal to a molar concentration of approximately 10 mM, with respect to the OECD guideline 476. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.

Vehicle / solvent:

- Solvent used: deionised water

- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10 % (v/v). This was chosen due to its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment: 14-18 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): approximately 7 days
- Selection time: 7-11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-18 days
- If a selective agent is used indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine (6-TG; 11 μg/mL), approximately 8 days (evaluation for viability) and approximately 9 ± 2 days (mutation analysis)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 0.7 to 1.2×10^7 24 h before treatment; The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
- Any supplementary information relevant to cytotoxicity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Rationale for test conditions:

The test conditions were selected according to OECD TG and results of the pre-experiment.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics. It was performed only for the positive controls, since all mean mutant frequencies of the groups treated with the test item were well within the 95 % confidence interval of our laboratory’s historical negative control data.
However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Possibility of evaporation from medium: not specified
- Water solubility: sufficiently soluble
- Precipitation and time of the determination: Neither precipitation nor phase separation was observed up to the highest tested concentration in the absence and in the presence of metabolic activation
- Definition of acceptable cells for analysis: Colonies with more than 50 cells were counted
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50 % or below was observed up to the highest concentration with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Please refer to “Any other information on results”.
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 0.7 to 1.2×10^7 were seeded 24 h before treatment. After treatment at least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultivated twice.
o Number of cells plated in selective and non-selective medium: selective: 4 - 5Χ10^5 cells/75 cm^² cell culture flask (5); non-selective: approx. 500 cells/25 cm^² flask (2)
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: : Please refer to “Any other information on results”.

HISTORICAL CONTROL DATA
- Positive historical control data: : Please refer to “Any other information on results”.
- Negative (solvent/vehicle) historical control data: : Please refer to “Any other information on results”.

Table 1 Summary of Results

				relative	relative	rel. adjusted	mutant	95%	statistical analysis*
	conc.	P/	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL	PS	mix	efficiency I	density	efficiency I	10^6 cells	interval	t-test	linear regression
				%	%	%
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4h treatment				Mean values of culture I and II
Solvent control with water			-	100.0	100.0	100.0	12.4	2.9 - 22.4
Positive control (EMS)	300.0		-	101.4	84.3	85.5	236.8	2.9 – 22.4	0.000^S
Test item	60.9	-	-	104.0	94.3	98.0	#
Test item	121.9	-	-	105.4	75.8	79.9	7.2	2.9 – 22.4	n.c.	0.769
Test item	243.8	-	-	88.2	94.4	83.2	13.5	2.9 – 22.4	n.c.
Test item	487.5	-	-	102.4	81.2	83.4	7.0	2.9 – 22.4	n.c.
Test item	975.0	-	-	95.4	95.7	90.9	7.4	2.9 – 22.4	n.c.
Test item	1950.0	-	-	89.7	102.5	91.6	10.2	2.9 – 22.44	n.c.
Solvent control with water			+	100.0	100.0	100.0	10.3	2.9 – 23.7
Positive control (DMBA)	2.3		+	89.6	81.5	73.3	143.7	2.9 – 23.7	0.000^S
Test item	60.9	-	+	85.3	86.4	73.5	#
Test item	121.9	-	+	91.8	92.9	85.1	6.9	2.9 – 23.7	n.c.	0.650
Test item	243.8	-	+	91.8	83.9	77.1	5.8	2.9 – 23.7	n.c.
Test item	487.5	-	+	88.6	83.4	74.2	7.7	2.9 – 23.7	n.c.
Test item	975.0	-	+	94.2	88.3	83.2	7.1	2.9 – 23.7	n.c.
Test item	1950.0	-	+	91.4	87.3	79.8	7.2	2.9 – 23.7	n.c.

* statistical analysis based on the mean values of culture I and II

P/PS = Precipitation/Phase separation at the end of treatment

S = significant trend (p < 0.05)

MF Mutant Frequency

# cultures not continued as a minimum of only four analyzable concentrations are required

 

Table 2 Cloning Efficiency I (Survival). culture I

	conc.	P/	S9	cells	number of colonies per flask			CE I	CE I	cells/mL	cell density	rel. adjusted
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	at 1st	%	CE I
				I/II	I	II	mean		%	subcultivation	of control	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Solvent control with water			-	518	398	436	417.0	0.8	100.0	1254000	100.0	100.0
Positive control with EMS	300.0		-	535	445	438	441.5	0.8	102.5	998000	79.6	81.6
Test item	60.9	-	-	509	421	396	408.5	0.8	99.7	1232000	98.2	97.9
Test item	121.9	-	-	519	439	445	442.0	0.9	105.8	884000	70.5	74.6
Test item	243.8	-	-	519	357	360	358.5	0.7	85.8	1256000	100.2	85.9
Test item	487.5	-	-	522	432	399	415.5	0.8	98.9	908000	72.4	71.6
Test item	975.0	-	-	509	375	316	345.5	0.7	84.3	1234000	98.4	83.0
Test item	1950.0	-	-	555	366	386	376.0	0.7	84.2	1344000	107.2	90.2
Solvent control with water			+	527	305	296	300.5	0.6	100.0	1422000	100.0	100.0
Positive control with DMBA	2.3		+	540	330	298	314.0	0.6	102.0	1188000	83.5	85.2
Test item	60.9	-	+	542	302	279	290.5	0.5	94.0	1192000	83.8	78.8
Test item	121.9	-	+	530	268	273	270.5	0.5	89.5	1432000	100.7	90.1
Test item	243.8	-	+	509	286	264	275.0	0.5	94.8	1234000	86.8	82.2
Test item	487.5	-	+	544	257	310	283.55	0.5	91.4	1318000	82.7	84.7
Test item	975.0	-	+	523	275	321	298.0	0.6	99.9	1268000	89.2	89.1
Test item	1950.0	-	+	535	274	282	278.0	0.5	91.1	1296000	91.1	83.1

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

 

Table 3 Cloning Efficiency II (Viability). culture I

	conc.	P/	S9	cells	number of colonies per flask			CE II	CE II	cells	cells
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	seeded	survived
				I/II	I	II	mean		%
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	537	397	412	404.5	0.8	100.0	457400	344541
Positive control with EMS	300.0		-	506	370	361	365.5	0.7	95.9	494400	357121
Test item	60.9	-	-	culture was not continued^#
Test item	121.9	-	-	520	453	428	440.5	0.8	112.5	387000	327834
Test item	243.8	-	-	524	329	347	338.0	0.6	85.6	388200	250404
Test item	487.5	-	-	569	353	336	344.5	0.6	80.4	400800	242664
Test item	975.0	-	-	534	413	381	397.0	0.7	98.7	471600	350609
Test item	1950.0	-	-	529	397	415	406.0	0.8	101.9	417600	320502
Solvent control with water			+	592	411	445	428.0	0.7	100.0	407400	294539
Positive control with DMBA	2.3		+	562	417	428	422.5	0.88	104.0	437400	328828
Test item	60.9	-	+	culture was not continued^#
Test item	121.9	-	+	502	429	448	438.5	0.9	120.8	403200	352198
Test item	243.8	-	+	574	420	454	437.0	0.8	105.3	407400	310163
Test item	487.5	-	+	548	442	417	429.5	0.8	108.4	404400	316952
Test item	975.0	-	+	528	455	423	439.0	0.8	115.0	436800	363173
Test item	1950.0	-	+	553	323	363	343.0	0.6	85.8	408300	253249

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

# culture was not continued as a minimum of only four analysable concentrations is required

 

Table 4 Mutagenicity data (Mutation rates). culture I

	conc.	P/	S9	number of colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6 cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	5	2	6	3	8	4.8	2.4	13.9
Positive control with EMS	300.0		-	68	78	66	82	65	71.8	7.7	201.1
Test item	60.9	-	-	culture was not continued^#
Test item	121.9	-	-	2	3	5	3	2	3.0	1.2	9.2
Test item	243.8	-	-	5	1	3	1	2	2.4	1.7	9.6
Test item	487.5	-	-	3	2	1	1	2	1.8	0.8	7.4
Test item	975.0	-	-	4	1	4	4	4	3.4	1.3	9.7
Test item	1950.0	-	-	2	3	1	4	2	2.4	1.1	7.5
Solvent control with water			+	3	5	1	8	1	3.6	3.0	12.2
Positive control with DMBA	2.3		+	46	43	51	47	46	46.6	2.9	141.7
Test item	60.9	-	+	culture was not continued^#
Test item	121.9	-	+	4	4	3	2	1	2.88	1.3	8.0
Test item	243.8	-	+	1	2	1	4	2	2.0	1.2	6.4
	487.5	-	+	3	3	1	2	2	2.2	0.8	6.9
Test item	975.0	-	+	2	2	4	3	3	2.8	0.8	7.7
Test item	1950.0	-	+	2	1	2	1	3	1.8	0.8	7.1

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

# culture was not continued as a minimum of only four analysable concentrations is required

 

Table 5 Cloning Efficiency I (Survival). culture II

	conc.	P/	S9	cells	number of colonies per flask			CE I	CE I	cells/mL	cell density	rel. adjusted
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	at 1st	%	CE I
				I/II	I	II	mean		%	subcultivation	of control	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Solvent control with water			-	561	440	431	435.5	0.8	100.0	1982000	100.0	100.0
Positive control with EMS	300.0		-	500	369	410	389.5	0.8	100.3	1766000	89.1	89.4
Test item	60.9	-	-	507	411	442	426.5	0.8	108.4	1792000	90.4	98.0
Test item	121.9	-	-	526	435	423	429.0	0.8	105.1	1608000	81.1	85.2
Test item	243.8	-	-	575	392	417	404.5	0.7	90.6	1758000	88.7	80.4
Test item	487.5	-	-	505	430	400	415.0	0.8	105.9	1784000	90.0	95.3
Test item	975.0	-	-	521	426	435	430.5	0.8	106.4	1842000	92.9	98.9
Test item	1950.0	-	-	548	419	391	405.0	0.7	95.2	1938000	97.8	93.1
Solvent control with water			+	509	337	308	322.5	0.6	100.0	1232000	100.0	100.0
Positive control with DMBA	2.3		+	533	267	255	261.0	0.5	77.3	980000	79.5	61.5
Test item	60.9	-	+	543	256	271	263.5	0.5	76.6	1096000	89.0	68.1
Test item	121.9		+	519	316	303	309.5	0.6	94.1	1048000	85.1	80.1
Test item	243.8	-	+	535	295	307	301.0	0.6	88.8	998000	81.0	71.9
Test item	487.5	-	+	522	273	295	284.0	0.5	85.9	914000	74.2	63.7
Test item	975.0	-	+	533	292	306	299.0	0.6	88.5	1076000	87.3	77.3
Test item	1950.0	-	+	509	301	290	295.5	0.6	91.6	1028000	83.4	76.5

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

 

Table 6 Cloning Efficiency II (Viability). culture II

	conc.	P/	S9	cells	number of colonies per flask			CE II	CE II	cells	cells
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	seeded	survived
				I/II	I	II	mean		%
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	553	368	404	386.0	0.7	100.0	555600	387815
Positive control with EMS	300.0		-	513	358	374	366.0	0.7	102.2	391800	279530
Test item	60.9	-	-	culture was not continued^#
Test item	121.9	-	-	523	451	477	464.0	0.9	127.1	523800	464710
Test item	243.8	-	-	553	303	325	314.0	0.6	81.3	444600	252449
Test item	487.5	-	-	512	280	324	302.0	0.6	84.5	406200	239595
Test item	975.0	-	-	564	357	350	353.5	0.6	89.8	444600	278663
Test item	1950.0	-	-	518	362	388	375.0	0.7	103.7	406200	294064
Solvent control with water			+	541	421	409	415.0	0.8	100.0	401400	307913
Positive control with DMBA	2.3		+	515	412	415	413.5	0.8	104.7	422400	339150
Test item	60.9	-	+	culture was not continued^#
Test item	121.9	-	+	514	402	395	398.5	0.8	101.1	402300	311900
Test item	243.8	-	+	556	441	418	429.5	0.8	100.7	394800	304976
Test item	487.5	-	+	500	423	405	414.0	0.8	107.9	400500	331614
Test item	975.0	-	+	550	411	427	419.0	0.8	99.3	402000	306251
Test item	1950.0	-	+	555	401	416	408.5	0.7	96.0	405000	298095

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

# culture was not continued as a minimum of only four analysable concentrations is required

 

Table 7 Mutagenicity data (Mutation rates). culture II

	conc.	P/	S9	number of colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6 cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	1	3	8	4	5	4.2	2.6	10.8
Positive control with EMS	300.0		-	88	69	72	72	80	76.2	7.8	272.6
Test item	60.9	-	-	culture was not continued^#
Test item	121.9	-	-	3	3	1	4	1	2.4	1.3	5.2
Test item	243.8	-	-	5	3	6	6	2	4.4	1.8	17.4
Test item	487.5	-	-	1	2	2	1	2	1.6	0.5	6.7
Test item	975.0	-	-	1	1	1	1	3	1.4	0.9	5.0
Test item	1950.0	-	-	6	3	3	3	4	3.8	1.3	12.9
Solvent control with water			+	2	1	3	2	5	2.6	1.5	8.4
Positive control with DMBA	2.3		+	40	53	46	57	51	49.4	6.6	145.7
Test item	60.9	-	+	culture was not continued^#
Test item	121.9	-	+	3	2	1	2	1	1.8	0.8	5.8
Test item	243.8	-	+	3	1	1	1	2	1.6	0.9	5.2
Test item	487.5	-	+	2	3	2	5	2	2.8	1.3	8.4
Test item	975.0	-	+	1	1	2	3	3	2.0	1.0	6.5
Test item	1950.0	-	+	2	4	2	1	2	2.2	1.1	7.4

P/PS = Precipitation/Phase separation at the beginning and at the end of treatment

# culture was not continued as a minimum of only four analysable concentrations is required

 

Table 8 Historical Laboratory Control Data

These values represent the historical control data from 2018-2020

Number of mutant colonies per 10^6 cells (mean values)
without metabolic activation (4 hours treatment time)
	Positive control EMS 300 µg/mL	Solvent control (medium. acetone. water. DMSO. ethanol. THF)
Range:	90 – 406.8	5.0 – 30.5
Mean value:	245.3	12.6
Standard deviation:	67.5	4.9
95% confidence interval	-	2.9 – 22.4
Number of studies:	83	83
with metabolic activation (4 hours treatment time)
	Positive control DMBA 2.3 µg/mL	Solvent control (medium. acetone. water. DMSO. ethanol. THF)
Range:	54.5 – 347.1	5.3 – 27.5
Mean value:	145.4	13.3
Standard deviation:	66.5	5.2
95% confidence interval	-	2.9 – 23.7
Number of studies:	83	83

 

The 95 % confidence interval is derived from the mean value plus/minus 2 times the standard deviation.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (1950 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. Based on the results of the pre-experiment the following concentrations were chosen for the main experiment in the presence and absence of metabolic activation: 60.9, 121.9, 243.8, 487.5, 975.0 and 1950.0 μg/mL. Neither precipitation nor phase separation occurred up the highest investigated concentration after four hours treatment. No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50 % (mean value of both parallel cultures) occurred up to the highest concentration. Consequently, the concentrations of 121.9 to 1950 μg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95 % confidence interval of the solvent historical control data. Linear regression analysis showed no statistically significant trend. The outcome of the experiment was clearly negative in the presence and in the absence of metabolic activation. The positive controls induced distinct increases in mutant frequencies, consistent with the laboratory’s historical positive control data, thus demonstrating the sensitivity of the test system and the efficacy of the S9 mix. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-27 to 2020-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: induced rat liver
- volume of S9 mix in the final culture medium: 0.5 mL S9 mix per plate
- quality controls of S9: not specified

Test concentrations with justification for top dose:

experiment 1 (dose-finding test): 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate
experiment 2 (main test): 5000, 2500, 1250, 625, and 313 µg/plate

Top dose according to guideline.

Vehicle / solvent:

- Solvent used: water for injection

- Justification for choice of solvent/vehicle:
Water for injection was selected as the solvent because the test substance is water-soluble and dissolves in water at a rate of 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2-AA); 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl (ICR-191)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Test conditions according to guideline were used.

Evaluation criteria:

A positive result was determined when the mean number of revertant colonies in the test substance-treated group increased more than 2-fold compared to the mean number of spontaneous revertant colonies (negative control value) and dose-response and reproducibility were observed, or when the mean number of revertant colonies increased more than 2-fold compared to the negative control value and reproducibility was observed in the dose-finding and main studies even when no clear dose-response was observed.

Statistics:

No statistical method was used for the determination.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No effects on pH reported.
- Data on osmolality: Not applicable
- Possibility of evaporation from medium: not specified
- Water solubility: Test item was sufficiently water soluble.
- Precipitation and time of the determination: No precipitation was detected.
- Definition of acceptable cells for analysis: Not applicable
- Other confounding effects: No futher effects observed.

RANGE-FINDING/SCREENING STUDIES: A dose-finding study was conducted and is used as experiment 1.

Ames test:
- Signs of toxicity : No cytotoxicity was detected.
- Please refer to "Any other information on results" for details.

HISTORICAL CONTROL DATA: Please refer to table 3.

Table 1: Experiment 1

Dose (µg/plate)	Mean number of revertant colonies/2 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Water	12	8	29	124	35
1.22	11	6	30	117	30
4.88	10	5	27	114	32
19.5	13	5	26	116	38
78.1	12	6	29	122	36
313	14	6	32	114	30
1250	13	8	25	115	30
5000	14	5	21	121	32
NaN3 (0.5)	521
AF-2 (0.01)				530	104
AF-2 (0.1)			541
ICR-191		1487
	Results with S9
Water	13	10	45	124	37
1.22	13	8	40	121	40
4.88	12	10	33	139	31
19.5	8	8	42	122	30
78.1	12	10	41	107	37
313	7	8	38	104	28
1250	10	10	39	140	33
5000	9	8	33	119	34
B(a)P (5)		74	335	1063
2-AA (2)	285
2-AA (10)					542

Table 2: Experiment 2

Dose (µg/plate)	Mean number of revertant colonies/2 replicate plates with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
Water	14	10	23	127	21
313	13	5	27	110	20
625	15	2	28	117	24
1250	16	5	22	134	20
2500	12	6	26	109	19
5000	15	3	28	106	18
NaN3 (0.5)	459
AF-2 (0.01)				494	98
AF-2 (0.1)			459
ICR-191		1311
	Results with S9
Water	14	8	38	136	27
313	9	9	35	126	22
625	13	7	42	108	22
1250	11	5	33	125	20
2500	12	7	42	122	21
5000	11	4	34	117	17
B(a)P (5)		64	397
2-AA (2)	295			295
2-AA (10)					633

Table 3: Historical Data (preincubation method)

Period: From June 1, 2020 to September 11, 2020

Strain	S9 mix		Management ranges		Mean	Management ranges		number of plates
			-3SD	-2SD		+2SD	+3SD
TA 100	-	Solvent control	74	92	128	165	183	234
		Positive control	413	455	540	624	66	228
	+	Solvent control	78	97	137	176	196	234
		Positive control	817	927	1146	1364	1485	228
TA1535	-	Solvent control	2	5	11	18	21	234
		Positive control	277	331	438	546	599	228
	+	Solvent control	1	4	11	18	21	234
		Positive control	214	256	340	424	466	228
WP2uvrA	-	Solvent control	6	12	24	36	42	234
		Positive control	56	72	102	133	148	228
	+	Solvent control	7	14	27	40	47	234
		Positive control	320	409	587	766	855	228
TA 98	-	Solvent control	4	11	24	38	45	234
		Positive control	430	464	532	600	634	228
	+	Solvent control	14	22	37	52	59	234
		Positive control	248	288	369	449	490	228
TA 1537	-	Solvent control	1	1	7	13	16	234
		Positive control	291	630	1310	1989	2329	228
	+	Solvent control	1	3	11	19	23	234
		Positive control	28	42	70	99	113	228

Conclusions:

The test item was determined to be non-mutagenic in a Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

To investigate whether the test item has the ability to induce gene mutations a bacterial reverse mutation assay according to OECD 471 was conducted. The preincubation method was used withSalmonella typhimurium (hereinafter referred to as S. typhimurium) TA100, TA1535, TA98, TA1537, and Escherichia coli (hereinafter referred to as E. coli) WP2uvrA under the conditions of metabolic activation and non-metabolic activation using S9. Water for injection was used as the solvent for the test substance. In order to establish the test dose, a dose-finding test was conducted with a total of seven doses of 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate diluted in six steps wit the ratio of 4.Results showed that there was no growth inhibition or precipitation of the test substance in any of the strains with or without metabolic activation. Therefore, the main test was conducted at a total of five doses of 5000, 2500, 1250, 625, and 313 µg/plate for all strains with and without metabolic activation. Regardless of the presence or absence of metabolic activation in both the dose-finding study and the main study, there was no increase of more than twice the negative control value in the number of revertant mutant colonies, in either the base-pair-substituted or the frameshifted strains, and no dose-response was observed. Based on the above test results, the test item was judged to have no gene mutagenic activity against bacteria under the test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test (reference 7.6.1-1)

To investigate whether the test item has the ability to induce gene mutations a bacterial reverse mutation assay according to OECD 471 was conducted. The preincubation method was used with Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA100, TA1535, TA98, TA1537, and Escherichia coli (hereinafter referred to as E. coli) WP2uvrAunder the conditions of metabolic activation and non-metabolic activation using S9. Water for injection was used as the solvent for the test substance. In order to establish the test dose, a dose-finding test was conducted with a total of seven doses of 5000, 1250, 313, 78.1, 19.5, 4.88, and 1.22 µg/plate diluted in six steps with the ratio of 4.Results showed that there was no growth inhibition or precipitation of the test substance in any of the strains with or without metabolic activation. Therefore, the main test was conducted at a total of five doses of 5000, 2500, 1250, 625, and 313 µg/plate for all strains with and without metabolic activation. Regardless of the presence or absence of metabolic activation in both the dose-finding study and the main study, there was no increase of more than twice the negative control value in the number of revertant mutant colonies, in either the base-pair-substituted or the frameshifted strains, and no dose-response was observed. Based on the above test results, the test item was judged to have no gene mutagenic activity against bacteria under the test conditions.

 

Micronucleus test (reference 7.6.1-2)

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in three independent experiments. Experiment I and II were conducted with and without metabolic activation (S9-mix) with exposure periods of 4 and 24 hours. Experiment III was conducted without metabolic activation with an exposure period of 24 hours. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1950 µg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The rationale for the dose selection is reported in section 3.5.1. In Experiments I, II and III in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment I in the presence of S9 mix, the value of 1.15% micronucleated cells after treatment with the highest applied concentration is statistically significantly increased in comparison to the solvent control but clearly within the 95% control limit of the historical control data (0.01 – 1.99% micronucleated cells). Dose dependency was observed using a trend test. In the confirmatory Experiment II in the presence of S9 mix none of these findings could be confirmed. Therefore, the findings in Experiment I can be considered as biologically irrelevant. In Experiment II and III in the absence of S9 mix, no relevant increases in micronucleated cells could be observed. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

 

HPRT assay (reference 7.6.1-3)

A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and the main experiment (1950 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. Based on the results of the pre-experiment the following concentrations were chosen for the main experiment in the presence and absence of metabolic activation: 60.9, 121.9, 243.8, 487.5, 975.0 and 1950.0 μg/mL. Neither precipitation nor phase separation occurred up the highest investigated concentration after four hours treatment. No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I (survival rate) below 50 % (mean value of both parallel cultures) occurred up to the highest concentration. Consequently, the concentrations of 121.9 to 1950 μg/mL were evaluated for mutagenicity in the presence and absence of metabolic activation. The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95 % confidence interval of the solvent historical control data. Linear regression analysis showed no statistically significant trend. The outcome of the experiment was clearly negative in the presence and in the absence of metabolic activation. The positive controls induced distinct increases in mutant frequencies, consistent with the laboratory’s historical positive control data, thus demonstrating the sensitivity of the test system and the efficacy of the S9 mix. EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.